The progesterone receptor (PR) is present in cells in two major isoforms, PR-A and PR-B. In the presence of a bound progestin ligand, such as progesterone, the PR is phosphorylated at specific sites, dimerizes, forms a complex with a number of different cellular elements (e.g., p300 and the steroid receptor coactivator), and binds to specific DNA sequences known as progesterone responsive elements (PREs) to initiate DNA transcription into RNA. The PR-ligand complex also attracts numerous other co-activators and co-repressors, which form the cellular elements which in turn transcribe particular genes. These PR complexes (also referred to as foci) can be visualized in the nuclei of cells which contain the progesterone receptor as fluorescent aggregates using immunohistofluorescence techniques and as dense and dark stained nuclear aggregates using the immunohistochemistry techniques described in this patent.
In premenopausal women, during the proliferative phase (the first part of the menstrual cycle) when estrogen is the dominant hormone and progesterone is minimally secreted, staining of normal endometrial cells for PR-A and PR-B (e.g., using immunofluorescent techniques and confocal microscopy) reveals a diffuse progesterone receptor nuclear staining pattern. In the secretory phase (the second part of the menstrual cycle) when progesterone is the dominant hormone, using the same immunofluorescent techniques and confocal microscopy, staining for PR-A and PR-B appears as readily detectable fluorescent nuclear foci.
RNA transcription inhibitors have been shown to prevent formation of PR foci, and 26S proteasome inhibitors have been shown to disrupt the PR nuclear foci. It is therefore believed that the presence of PR foci in cells corresponds to active transcriptional complexes, and indicates the activation of the PR and subsequent gene expression. Conversely, diffuse nuclear staining or the absence of PR foci indicates the presence of PR which is transcriptionally inactive. Upon exposure of normal breast and endometrium tissues (which are physiologically responsive to progesterone) to progestin ligands, a change from a diffuse nuclear staining pattern to focal subnuclear structures can be observed, indicating the activation of the progesterone receptor.
Whereas estrogens are mitogenic (e.g., cause cellular proliferation) for normal breast epithelial and endometrial cells, the effects of progestins are more complex. In the endometrium, progestins inhibit estrogen-induced cell cycle progression early in the G1 phase, whereas in the breast progestins may both stimulate and inhibit proliferation. In normal breast tissue biopsies it has been shown that proliferative activity is stimulated by progesterone (Am J Obstet Gynecol, 1997). This complexity has led to confounding experimental observations in breast cancer. For example, progestogens appear to have a direct proliferative effect on breast cancer cell in vitro when phenol red-free media is used. H. J. Kloosterboer, J. Steroid Biochem. Molec. Biol. Vol. 49, No. 4-6, pp. 311-318, 1994. However, when the same contraceptive progestogens that induced proliferation in breast cancer cell lines were studied in an estrogen-dependent DMBA rat breast cancer model, these progestogens inhibited tumor progression. Id. It has been shown recently that many such in vitro experimental models are inadequate. See, e.g., Lange C. et al. Progesterone Receptor Action: Translating Studies in Breast Cancer Models to Clinical Insights. Chapter 7 in Innovative Endocrinology of Cancer; 94-111 (2010). While progesterone-induced proliferation has been shown in these experimental models, the majority of proliferating cells were not expressing the PR. Thus, these models do not necessarily predict the efficacy of treatment with antiprogestins.
Malignant cells also exhibit nuclear PR foci, but they are different in size and composition from the foci of normal cells. PR foci observed in cancer indicate a specific role for the PR which is pertinent to the malignant nature of the cells. For example, the genes activated by the PR in malignant (cancer) breast cells are different than the genes activated by the PR in normal breast cells; in endometrial cancers PR foci, but not PR levels, are associated with malignant characteristics; foci in cancer cells are larger, which may be due to alterations in the chromatin remodeling which are common in cancer, and; PR foci in breast cancer are observed regardless of hormonal status (e.g., in the presence and absence of circulating progesterone in premenopausal and post-menopausal women respectively). PR foci have been observed (e.g., using immunofluorescent techniques and confocal microscopy) in the tumor cells of approximately 50% of PR-receptor positive human breast cancer biopsies. Other patient's tumor samples exhibited a diffuse PR nuclear staining pattern in the tumor cells using immunofluorescent techniques and confocal microscopy, indicative of a non-activated or non-functional form of the PR.
The majority of breast cancers can be treated with hormonal treatments (i.e., anti-estrogens or aromatase inhibitors), which are currently some of the most effective medications used in breast cancer therapy. Hormonal treatment is usually indicated based on the identification of hormone receptors within the cancer cells. Onapristone (ONA) is an anti-progestin drug which was originally developed for contraceptive use. However, it has demonstrated substantial activity in advanced breast cancer, with a 10% response rate in a study of 101 poor prognosis patients with breast cancer in whom prior hormonal therapy had failed (e.g., breast cancer progressed despite the patient receiving the antiestrogen tamoxifen). In a small breast cancer study using ONA as a first line hormone treatment, ONA produced a 56% objective response rate, an efficacy in the upper range of the best available treatments in this disease. ONA binds to the PR, does not induce PR phosphorylation and does not allow the PR to dimerize. The PR-ONA complex binds weakly, or not at all, to its target DNA segment and therefore does not activate the chromatin remodeling which is a necessary process for DNA transcription. In in vitro systems, ONA has been shown to reverse the PR nuclear aggregates produced by binding of an artificial ligand to the PR. Gene activation studies have consistently shown that, while progestins and other anti-progestins activate progesterone responsive genes, ONA has minimal activation (i.e., 3 genes).
In addition, ONA is a pure PR antagonist at concentrations which can be physiologically achieved. ONA does not interfere with other steroid receptors and does not increase estrogen secretion in human subjects, which is an undesirable side-effect for breast cancer therapy exhibited by other anti-progestins such as mifepristone.
While onapristone has previously been investigated as a potential therapeutic agent for breast cancer, its development was stopped due to toxicity concerns. Robertson et al., Onapristone, a Progesterone Receptor Antagonist, as First-line Therapy in Primary Breast Cancer European J. of Cancer 35(2) 214-218 (1999). It is important to identify the subset of the patients with tumors most likely to respond and equally as important to identify the subset of the patients with tumors least likely to respond to treatment with ONA and other anti-progestins. Identifying these subsets of patients will allow those patients with APF access to a potentially effective cancer treatment and will avoid exposing patients with those cancers for which ONA or other anti-progestins may not provide benefit to unnecessary toxicity.
Currently, only the presence or absence of the estrogen or progesterone receptor is considered when making therapeutic decisions on whether to use an endocrine treatment in certain cancers (e.g., breast cancer). Accordingly, conventional assays for PR classify the tumors from patients with cancer into two categories: PR-positive or PR-negative. One type of assay quantitates the amount of PR per total protein of the cell. These methods can be automated and are quantitative, but are not satisfactory with respect to accuracy, sensitivity and analysis of cellular subnuclear receptor structures. A second type of assay includes immunohistochemical methods using formalin fixed tissue specimens and fluorescent or chromophore labeled monoclonal antibodies targeting the receptor (either an antibody for each of PR-A and PR-B, or a single antibody that recognizes both). With immunohistochemical methods, any microscopically detectable nuclear staining reaction in more than a certain percentage of cells (typically ≧1%), is reported as being PR positive as per professional society guidelines. Typically, a clinical cut off of ≧10% ER or PR positive cells is used to make therapeutic decisions regarding the use of anti-hormone treatments. No consideration is given to the pattern of cellular or nuclear staining. Relative staining intensity (i.e., low, medium, or high) is also use as a qualitative measure of hormone receptor positivity. This second type of assay is more labor intensive and it is not standardized. Typically, low magnification microscopic examination is used for the IHC analysis to identify the presence of the hormone receptor (either estrogen receptor (ER) or PR). Using conventional methods, no analysis of cellular distribution is done other then an estimate of the percentage of the tumor cells expressing the identified hormone receptor. Analysis of the subnuclear distribution pattern of the PR requires high powered microscopy. In contrast, high powered microscopy is not needed for standard IHC determination of hormone receptors in tumor tissue. These conventional methods of hormone receptor determination are thus unable to provide information regarding subnuclear PR distribution.
Progestins have complex actions in the breast and other hormone sensitive tissues by targeting distinct cells and having indirect effects on cells not expressing the PR. PR foci complexes are not qualitatively the same in normal tissue and cancerous tissue, and they do not necessarily activate the same progesterone receptor associated genes. Available clinical data does not fully support the position that conventional techniques for identifying hormone receptor positive cells are predictive of anti-hormone efficacy, whether it be for anti-estrogen or anti-progestin directed treatments. Currently, the decision to utilize a hormone treatment (e.g., antiestrogens or aromatase inhibitors) for patients with breast cancer and other hormone sensitive tumors is based on the simple presence of hormone-receptors in tumor samples. The presence of hormone receptors (ER or PR) does not fully predict for response to hormone treatment, as only 50-60% of hormone-receptor positive tumor cases are expected to benefit from treatment.
There is a need for a consistent method for predicting the efficacy of ONA and other anti-progestins with respect to heterogeneous “naturally occurring” tumors. Further, there is a need for an assay which is predictive of therapeutic efficacy of ONA and other anti-progestins against the cancers in individual patients.